Temperature effects on the fatty acid (FA) profiles of phytoplankton, major primary producers in the ocean, have been widely studied due to their importance as industrial feedstocks and to their indispensable role as global producers of long-chain, polyunsaturated FA (PUFA), including omega-3 (ω3) FA required by organisms at higher trophic levels. The latter is of global ecological concern for marine food webs, as some evidence suggests an ongoing decline in global marine-derived ω3 FA due to both a global decline in phytoplankton abundance and to a physiological reduction in ω3 production by phytoplankton as temperatures rise. Here, we examined both short-term (physiological) and long-term (evolutionary) responses of FA profiles to temperature by comparing FA thermal reaction norms of the marine diatom Thalassiosira pseudonana after ~500 generations (ca. 2.5 years) of experimental evolution at low (16°C) and high (31°C) temperatures. We showed that thermal reaction norms for some key FA classes evolved rapidly in response to temperature selection, often in ways contrary to our predictions based on prior research. Notably, 31°C-selected populations showed higher PUFA percentages (including ω3 FA) than 16°C-selected populations at the highest assay temperature (31°C, above T. pseudonana’s optimum temperature for population growth), suggesting that high-temperature selection led to an evolved ability to sustain high PUFA production at high temperatures. Rapid evolution may therefore mitigate some of the decline in global phytoplankton-derived ω3 FA production predicted by recent studies. Beyond its implications for marine food webs, knowledge of the effects of temperature on fatty acid profiles is of fundamental importance to our understanding of the mechanistic causes and consequences of thermal adaptation.